The nature and causes of cancer are not entirely understood. In general, it is believed that cancer may be caused when one or more cells are genetically transformed in some manner which hinders or removes the normal limitations on the reproductive capacity of the cells [1, 2]. If the transformed genetic structure is inherited by descendant cells, the cells undergo uncontrolled proliferation, which is characteristic of malignant tumors.
The biochemical substances and processes involved in genetic replication are very complex, and relatively minor variations may result in substantial or extreme alterations in the descendant cells [3, 4, 5].
In general, tumor formation is believed to be a two-stage process. The initial event of carcinogenesis is called "initiation," which involves the transformation of a cell into a "pre-cancerous" cell. The following stage is called "promotion," which involves the proliferation and possible evolution of the transformed cell, thereby creating a tumor. Numerous substances and processes are believed to enhance the initiation or the promotion of cancerous cells. These substances and processes are usually referred to as "carcinogens" and "tumor promoters," depending upon whether their predominant effect relates to the initiation or the promotion of tumors.
In general, a carcinogen is a substance or process which, when exposed to cells, increases the rate or probability of genetic transformation of the cells or their descendants, resulting in the emergence of cells capable of forming tumors [6, 7]. Carcinogens are believed to include viruses [8], chemical substances [9], and radiation [10].
A tumor promoter is a substance which, when exposed to cells in combination with one or more carcinogens, significantly increases the size or incidence of tumors [11, 12]. To determine whether a suspected tumor promoter is active mainly during the promotion stage, the substance may be administered to an animal without additional administration of a carcinogen; a low incidence of tumors indicates a low level of carcinogenicity.
Both carcinogens and tumor promoters may be somewhat cytotoxic; i.e., at sufficiently high concentrations, they may kill cells directly, thereby preventing them from reproducing. In addition, carcinogens may have some degree of tumor-promoting capacity, and tumor promoters may have some degree of carcinogenicity.
A common procedure for measuring the tumor-promoting characteristic of a substance or process is to administer the substance or process to an animal in combination with a carcinogen that has a known level of carcinogenicity. For example, in one common experimental protocol [13], the backs of numerous mice are shaved, and the mice are divided into two categories, one of which serves as a control group. A predetermined quantity of a known carcinogen is administered to the backs of the control-group mice. This same amount of carcinogen, plus a predetermined quantity of a suspected tumor promoter, are administered to the backs of the other group of mice. If the doubly-exposed mice develop a significantly higher incidence of tumors on their backs, the suspected substance is regarded as a tumor promoter. For example, a class of substances known as phorbol esters is known to contain several substances that are tumor promoters. In particular, 12-O-tetradecanoylphorbol--13-acetate (TPA) has been shown by mouse skin assays to be a very potent tumor promoter. When a relatively low dose of dimethylcholantren was applied alone to mouse skin, the incidence of tumors was approximately six percent. However, when both TPA and the same dose of dimethylcholantren were applied to mouse skin, the incidence of tumors increased to nearly 100 percent [14].
This experimental procedure suffers several limitations and drawbacks, including the following:
1. It is time-consuming. It may require several weeks or months for all incipient tumors to develop, depending upon the carcinogenicity of the substances administered.
2. It is expensive. Even the simplest and most common laboratory animals, such as common mice, are relatively expensive to procure and properly feed and care for. Special lines of mice with various desired characteristics are often extremely expensive. Research involving higher mammals such as dogs, pigs, and primates is often necessary, and is correspondingly more expensive.
3. Research involving animals is less reproducible, and is more subject to extraneous variables that cannot be entirely eliminated or predicted. Since only a limited number of animals may be used for a given experiment, small variations (e.g., deaths because of extraneous factors) can cause significant variations in the results. By contrast, millions of cells may be used quickly and inexpensively in a cellular assay.
4. Research involving animals requires far more space than research involving cells. In biological research laboratories, space is valuable and expensive.
For these reasons, it is very desirable and useful to measure the tumor promoting potential of a substance or process by assays involving cultures of cells, rather than animals.
A variety of cellular assays involving TPA and other tumor promoters have shown that tumor promoters induce a wide-range of metabolic effects, including stimulation of macromolecular metabolism and cell growth [17], alteration of various plasma membrane functions such as phospholipid metabolism [18] and sugar transport [19], suppression or enhancement of terminal differentiation [20], induction of viral antigens, [21] and altered cellular morphology [22]. However, none of these assays is necessarily directly related to genetic transformation of cells; in addition, other assays have shown that TPA does not display mutagenic activity in conventional mutation assays [23]. Therefore, such assays may not provide a reliable indication of whether a substance or process can promote tumors or other genetic transformation.
A different type of assay has been separately developed to determine gene amplification. This assay has been used to study the causes and characteristics of cellular resistance to drugs. One such assay involves methotrexate (MTX), a cytotoxic drug. MTX kills cells by binding very tightly to an enzyme, dihydrofolate reductase (DHFR). A single molecule of DHFR enzyme is inactivated by a single molecule of MTX. Since DHFR is essential to the metabolism and reproduction of a cell, a cell that contains a normal quantity of DHFR molecules may be killed by a predetermined quantity of MTX. However, some cells produce higher quantities of DHFR than do other cells. These cells may have enough DHFR to resist and survive exposure to a given concentration of MTX, and to reproduce. It has been shown that MTX resistance, which is due to abnormally high quantities of DHFR, is primarily due to amplification of the DHFR locus of the DNA molecule. In other words, in MTX-resistant cells, the portion of DNA that codes for the production of the DHFR enzyme is repeated an abnormally high number of times either within the chromosome or as extra-chromosomal DNA [24].
Methotrexate is not the only selective agent that can be used to assess gene amplification. For example, several heavy metals such as cadmium are cytotoxic. These metals can be bound and inactivated by certain proteins, such as metallothionein. Therefore, cells that have an abnormally high quantity of metallothionein are more likely to survive exposure to heavy metals such as cadmium. The presence of abnormally high quantities of metallothionein or similar enzymes is indicated by resistance to cadmium or similar selective agents. The correlation of such resistance to gene amplification, rather than to induced transcription or translation, has been demonstrated [25]. Similarly, the addition of phosphonacetyl-L-aspartate to a cell culture kills cells that do not have an abnormally high quantity of loci that code for a multifunctional protein referred to as CAD [26]. In general, an enzyme or other biological substance that is bound, inactivated, destroyed, or otherwise altered by a drug or other substance is regarded as a "target" of that substance.
Mutations and genetic transformations that are induced by carcinogens or mutagens are presumed to occur randomly within the cellular genome. Therefore, amplification of a single genetic locus, such as the DHFR locus, is presumed to be representative of amplification of numerous other loci as well. The number of copies of a certain type of locus within a cell may be determined through the use of radioactive "probes" by using a process known as "dot hybridization" [27]. This assay can be used to support the interpretation that an abnormally high quantity of an enzyme is caused by gene amplification, rather than by activation of transcription or translation.